Tenofovir versus entecavir on recurrence and mortality of hepatitis B virus-related hepatocellular carcinoma after curative therapy.

BACKGROUND: Tenofovir disoproxil fumarate (TDF) and entecavir (ETV) reduce the risk of hepatocellular carcinoma (HCC) in patients of hepatitis B. This study compared the difference between ETV and TDF on risk of HCC recurrence and mortality in patients with HBV-related HCC after curative intent treatment. METHODS: Patients with HBV-related HCC who received HCC treatment (surgery or radiofrequency ablation [RFA]) and underwent long-term ETV or TDF therapy were retrospectively included. Baseline characteristics including age, sex, antiviral therapy, liver reserve, HCC stages, pathology reports and treatment modality were obtained. The risk of tumor recurrence, all-cause mortality, HCC-related mortality, and liver function were compared. RESULTS: We identified 390 HBV-related HCC patients with curative intent treatment for HCC and treated with ETV (n = 328) or TDF (n = 62) between January 2011 and December 2020. The median age was 60 years, and 90.7% patients were males. After a median follow-up of 29 months, 186 patients developed recurrent HCC and 111 died. The baseline characteristics were comparable except more ALBI grade 3 patients in TDF group (76% vs. 48%, P < 0.001). Compared to ETV group, TDF users had lower all-cause mortality (adjusted hazard ratio [aHR]: 0.38, P = 0.003), and HCC-related mortality (aHR: 0.23, P = 0.005). Lower recurrence rate was noticed in TDF users after inverse probability of treatment weighting (IPTW). TDF users had improved ALBI grade and FIB-4 index compared with ETV groups. CONCLUSIONS: TDF therapy is associated with a reduced risk of HCC-related outcomes among patients with HBV-related HCC after curative intent treatment compared with ETV usage.

Sequencing Ultrarare Targets with Compound Nucleic Acid Cytometry.

Targeted sequencing enables sensitive and cost-effective analysis by focusing resources on molecules of interest. Existing methods, however, are limited in enrichment power and target capture length. Here, we present a novel method that uses compound nucleic acid cytometry to achieve million-fold enrichments of molecules >10 kbp in length using minimal prior target information. We demonstrate the approach by sequencing HIV proviruses in infected individuals. Our method is useful for rare target sequencing in research and clinical applications, including for identifying cancer-associated mutations or sequencing viruses infecting cells.

High diversity droplet microfluidic libraries generated with a commercial liquid spotter.

Droplet libraries consisting of many reagents encapsulated in separate droplets are necessary for applications of microfluidics, including combinatorial chemical synthesis, DNA-encoded libraries, and massively multiplexed PCR. However, existing approaches for generating them are laborious and impractical. Here, we describe an automated approach using a commercial array spotter. The approach can controllably emulsify hundreds of different reagents in a fraction of the time of manual operation of a microfluidic device, and without any user intervention. We demonstrate that the droplets produced by the spotter are similarly uniform to those produced by microfluidics and automate the generation of a ~ 2 mL emulsion containing 192 different reagents in ~ 4 h. The ease with which it can generate high diversity droplet libraries should make combinatorial applications more feasible in droplet microfluidics. Moreover, the instrument serves as an automated droplet generator, allowing execution of droplet reactions without microfluidic expertise.

Coordination among tertiary base pairs results in an efficient frameshift-stimulating RNA pseudoknot.

Frameshifting is an essential process that regulates protein synthesis in many viruses. The ribosome may slip backward when encountering a frameshift motif on the messenger RNA, which usually contains a pseudoknot structure involving tertiary base pair interactions. Due to the lack of detailed molecular explanations, previous studies investigating which features of the pseudoknot are important to stimulate frameshifting have presented diverse conclusions. Here we constructed a bimolecular pseudoknot to dissect the interior tertiary base pairs and used single-molecule approaches to assess the structure targeted by ribosomes. We found that the first ribosome target stem was resistant to unwinding when the neighboring loop was confined along the stem; such constrained conformation was dependent on the presence of consecutive adenosines in this loop. Mutations that disrupted the distal base triples abolished all remaining tertiary base pairs. Changes in frameshifting efficiency correlated with the stem unwinding resistance. Our results demonstrate that various tertiary base pairs are coordinated inside a highly efficient frameshift-stimulating RNA pseudoknot and suggest a mechanism by which mechanical resistance of the pseudoknot may persistently act on translocating ribosomes.

Quantitative analysis of ligand-EGFR interactions: a platform for screening targeting molecules.

Epidermal growth factor receptor (EGFR) is often constitutively stimulated in many cancers owing to the binding of ligands such as epidermal growth factor (EGF). Therefore, it is necessary to investigate the interaction between EGFR and its targeting biomolecules. The main aim of this study was to estimate the binding affinity and adhesion force of two targeting molecules, anti-EGFR monoclonal antibody (mAb LA1) and the peptide GE11 (YHWYGYTPQNVI), with respect to EGFR and to compare these values with those obtained for the ligand, EGF. Surface plasmon resonance (SPR) was used to determine the equilibrium dissociation constant (KD) for evaluating the binding affinity. Atomic force microscopy (AFM) was performed to estimate the adhesion force. In the case of EGFR, the KD of EGF, GE11, and mAb LA1 were 1.77 × 10-7, 4.59 × 10-4 and 2.07 × 10-9, respectively, indicating that the binding affinity of mAb LA1 to EGFR was higher than that of EGF, while the binding affinity of GE11 to EGFR was the lowest among the three molecules. The adhesion force between EGFR and mAb LA1 was 210.99 pN, which is higher than that observed for EGF (209.41 pN), while the adhesion force between GE11 and EGFR was the lowest (59.51 pN). These results suggest that mAb LA1 binds to EGFR with higher binding affinity than EGF and GE11. Moreover, the adhesion force between mAb LA1 and EGFR was greater than that observed for EGF and GE11. The SPR and AFM experiments confirmed the interaction between the receptor and targeting molecules. The results of this study might aid the screening of ligands for receptor targeting and drug delivery.

Neurogenic differentiation of dental pulp stem cells to neuron-like cells in dopaminergic and motor neuronal inductive media.

BACKGROUND/PURPOSE: Dental pulp stem cells (DPSCs) have been proposed as a promising source of stem cells in nerve regeneration due to their close embryonic origin and ease of harvest. The aim of this study was to evaluate the efficacy of dopaminergic and motor neuronal inductive media on transdifferentiation of human DPSCs (hDPSCs) into neuron-like cells. METHODS: Isolation, cultivation, and identification of hDPSCs were performed with morphological analyses and flow cytometry. The proliferation potential of DPSCs was evaluated with an XTT [(2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)] assay. Media for the induction of dopaminergic and spinal motor neuronal differentiation were prepared. The efficacy of neural induction was evaluated by detecting the expression of neuron cell-specific cell markers in DPSCs by immunocytochemistry and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: In the XTT assay, there was a 2.6- or 2-fold decrease in DPSCs cultured in dopaminergic or motor neuronal inductive media, respectively. The proportions of ßIII-tubulin (ßIII-tub), glial fibrillary acidic protein (GFAP), and oligodendrocyte (O1)-positive cells were significantly higher in DPSCs cultured in both neuronal inductive media compared with those cultured in control media. Furthermore, hDPSC-derived dopaminergic and spinal motor neuron cells after induction expressed a higher density of neuron cell markers than those before induction. CONCLUSION: These findings suggest that in response to the neuronal inductive stimuli, a greater proportion of DPSCs stop proliferation and acquire a phenotype resembling mature neurons. Such neural crest-derived adult DPSCs may provide an alternative stem cell source for therapy-based treatments of neuronal disorders and injury.

Prototype of biliary drug-eluting stent with photodynamic and chemotherapy using electrospinning.

BACKGROUND: The combination of biliary stent with photodynamic and chemotherapy seemed to be a beneficial palliative treatment of unresectable cholangiocarcinoma. However, by intravenous delivery to the target tumor the distribution of the drug had its limitations and caused serious side effect on non-target organs. Therefore, in this study, we are going to develop a localized eluting stent, named PDT-chemo stent, covered with gemcitabine (GEM) and hematoporphyrin (HP). METHODS: The prototype of PDT-chemo stent was made through electrospinning and electrospraying dual-processes with an electrical charge to cover the stent with a drug-storing membrane from polymer liquid. The design of prototype used PU as the material of the backing layer, and PCL/PEG blends in different molar ratio of 9:1 and of 1:4 were used in two drug-storing layers with GEM and HP loaded respectively. RESULTS: The optical microscopy revealed that the backing layer was formed in fine fibers from electrospinning, while drug-storing layers, attributed to the droplets from electrospraying process. The covered membrane, the morphology of which was observed by scanning electron microscopy (SEM), covered the stent surface homogeneously without crack appearances. The GEM had almost 100% of electrosprayed efficiency than 70% HP loaded on the covered membrane due to the different solubility of drug in PEG/PCL blends. Drug release study confirmed the two-phased drug release pattern by regulating in different molar ratio of PEG/PCL blends polymer. CONCLUSIONS: The result proves that the PDT-chemo stent is composed of a first burst-releasing phase from HP and a later slow-releasing phase from GEM eluting. This two-phase of drug eluting stent may provide a new prospect of localized and controlled release treatment for cholangiocarcinoma disease.